Table Of ContentAmer. Malac. Bull. 25: 35-41 (2008)
Methods of sample preparation of radula epithelial tissue in chitons (Mollusca:
Polyplacophora)
Jeremy A. Shaw\ David J. Macey\ Peta L. Clode^, Lesley R. Brooker^, Richard I. Webb‘S,
Edward Stockdale^, and Rachel M. Binks*
J.
* School ofBiological Sciences and Biotechnology, Murdoch University, Murdoch, Western Australia, 6150,
Australia,[email protected]
^Centre for Microscopy, Characterisation and Analysis, The University ofWestern Australia, Crawley, Western Australia, 6009,
Australia.
^ Faculty ofScience, Health and Education, University ofthe Sunshine Coast, Maroochydore, District ofCairns, Queensland, 4558,
Australia.
^Centre for Microscopy and Microanalysis, Department for Microbiology and Parasitology, University ofQueensland, St. Lucia,
Queensland, 4072, Australia.
Abstract: A glutaraldehyde fixative developed for preserving the radula superior epithelium of the adult chiton Acanthopleura hirtosa
(Blainville, 1825), was used in conjunction with conventional and microwave-assisted sample processing to produce high c]uality tissue
preservation forlight and electron microscopy. In addition, high-pressure freezing (HPF) and cryo-substitution were used to fixthe radula
tissue ofjuvenile specimens. Microwave-assisted fixation was preferred to conventional bench-top techniques due to the superior preser-
vation offine cell structure together with reduced processing times and chemical exposure. Although restricted to very small (<200 pm)
samples, the quality ofjuvenile radulae processed by HPF was excellent. The improvements in tissue preservation using microwave and
cryo-preservation techniques are therefore critical for obtaining accurate ultrastructural information on the radula in marine molluscs. In
particular, these findings highlight additional processing options available for the study ofcellular structures in biomineralizing tissues.
Keywords: microwave, high-pressure freezing, chemical fixation, cryo-fixation, biomineralization
The radula has been the focus ofnumerous studies over transport of ions to the tooth cusps are poorly understood.
many decades, with its intricate and varied design used to The elemental precursors for biomineralization are thought
elucidate aspects of molluscan ecology, biology, and tax- to be delivered to the teeth by the overlying, superior epi-
onomy (Fretter and Graham 1962, Runham 1963, Steneck thelial tissue, which surrounds the cusps during all stages of
and Watling 1982, Padilla 1985, Scheltema 1988, Salvini- development (Nesson and Lowenstam 1985) (Fig. 1)- The
Plawen 1990). In addition, the radulae of polyplacophoran superior epithelium and teeth are encapsulated within the
and patellid gastropods have received particular attention as radularsheath, the inferior epithelium, and the radula mem-
a result oftheir unique abilityto harden their teeth with iron brane with the whole structure resembling a tube open only
and other biominerals (Mann et al. 1986, Lowenstam and alongthedorsalsurface (Fig. 1). Histological investigation of
Weiner 1989, Webb et al. 1989). epithelial tissue is difficult due to the complex composition
The chiton radula represents an excellent example of and structure ofthe radula, which contains both hard min-
matrix-mediated biomineralization, where minerals are eralized structures and cartilaginous membranes in close
formed in a highly organized manner within the framework proximity to cellular material.
of an organic matrix (Simkiss and Wilbur 1989, Watabe Chemical fixatives such as glutaraldehyde buffered in
1990, Mann 2001, Weiner and Addadi 2002). While consid- filtered seawater are commonly used for preserving marine
erable progress has been made in elucidating the general organisms,wheretheosmotic pressureofthesolution actsto
structural organization of minerals deposited within the mimic that of the animal, thereby reducing swelling or
tooth matrix and the sequence in which they are deposited shrinkage ofthe tissues (Dykstra and Reuss 2003). However,
(Lowenstam 1962, Kim et al. 1989, Macey et al. 1994, fixation often gives rise to variations in the ultrastructural
Brooker et al. 2006), the mechanisms involved in cellular morphology of organelles (Hayat 2000), and it is therefore
From the symposium “Advances in Chiton Research” presented at the joint meetingoftheAmerican Malacological Societyand Western
Society ofMalacologists, held 29 luly to 3 August 2006 in Seattle, Washington.
35
36 AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008
preferable to utilize several techniques
to acquire comparative information.
Microwave-assisted fixation and cryo-
preservation techniques (McDonald et
al. 2007, Webster 2007), nowcommon
in laboratories, provide two additional
means of obtaining such comparative
information. The main advantage of
microwave protocols is a dramatic re-
duction in sample processing times
(<4 hours), while at least maintaining,
if not improving, fixation quality over
conventional methods (Giberson and
Demaree 1999, Laboux et ah 2004). Figure 1. Radula apparatus of the chiton Acanthopleura hirtosa (adult) in (A) transverse
Excellent tissue preservation can also section and (B) longitudinal section exhibiting various hard and soft tissue components.
be attained using cryo-preservation Abbreviations: se,superiorepithelium; ie, inferiorepithelium; tc, tooth cusp; ts, tooth stylus;
techniques, which achieve vitreous ice rm, radula membrane; rs, radula sheath. Scale bars = 200 pm.
formation in samples and therebypre-
vent ice crystal damage. However, due
to difficulties associated with heat dissipation, only very MATERIALS AND METHODS
small samples (<200 pm) can be frozen successfully (Wilson
et al. 1998, Sawaguchi et al. 2005). Conventional and microwave-assisted chemical fixation
To improve our current techniques and better under- and cryo-fixation techniques were utilized for the prepara-
stand the detailed cellular structure of the superior epithe- tion ofepithelial tissues for light microscopy (LM) or trans-
lium in chiton and limpet teeth, we investigated alternative mission electron microscopy (TEM). Adult and juvenile
fixation methods. Determining the precise structure ofthese specimens ofthe chitonAcanthopleura hirtosa (mean animal
cells will assist in elucidating their function and the mecha- length ~4 cm and -0.8 cm, respectively) were collected from
nisms involved in the transport of ions into the teeth, a intertidal limestone at Woodman Point within the Perth
fundamental obstacle to our wider understanding ofthe ini- metropolitan area ofWestern Australia (32°08'S, 115°44'E).
tial phase of biomineralization. The aim of this study is to Incisions were made along both pallial grooves from the anus
compare three fixation methodologies (conventional chemi- towards the head, thereby freeing the foot, visceral mass,
cal, microwave-assisted, and low temperature), regarding and buccal mass as a single entity from the shell plates and
preservation of the superior epithelial tissues of the chiton girdle. The visceral mass was then carefullyremoved to expose
Acanthopleura hirtosa (Blainville, 1825) for both light and the radula sac. Dissections were performed as quickly as pos-
electron microscopy. sible to reduce the deterioration of radula epithelial tissue.
Table 1. Conventional and microwave-assisted processing schedules for the radula epithelial tissues ofadult Acanthopleurahirtosa.
Conventional Microwave
Step Medium Concentration (%) times Microwave times wattage (W)
Fixation glutaraldehyde 2.5* 24 2x(2°U2°fV2°")(min)(v) 80
(buffer A)
Rinse buffer A 15 min x4 40 sec (v) 250
Post-fixation OSO4 (buffer B) U 2h 2x(2“72°^V2°")(min)(v) 80
Rinse buffer B 15 min x4 40 sec (v) 250
Dehydration acetone 10, 30, 50*, 75*, 90*, 100* 15 min x2 each 40 sec each (100 x2) 250
Infiltration Spurr’s resin 5, 10, 20, 40, 50, 60, 75, 80, 90, 100* 8-12 h each 3 min each (100 x2) (v) 250
Polymerization Spurr’s resin 100* conventional oven 60°C overnight
Note: Concentrations in bold represent the microwave schedule, concentrations not in bold represent the conventional schedule, and * represents
concentrations used in both schedules.
(v) = steps undertaken in vacuum
denotes magnetron (irradiation) cycle
SAMPLE PREPARATION OP RADULA EPITHELIUM 37
Table 2. Conventional and microwave-assisted processing schedules for the radula epithelial tissues ofjuvenile Acauthopleura hirtosa.
Conventional Microwave
Step Medium Concentration (%) times Microwave times wattage (W)
Fixation glutaraldehyde 2.5* 24 h 2x{2""/2""/2'’”) 80
(buffer A) (min) (v)
Rinse buffer A 10 min x4 40 sec (v) 250
Post-fixation OSO4 (buffer B) 1* 1 h 2x(2"'V2'’'V2"") 80
(min) (v)
Rinse buffer B 10 min x4 40 sec (v) 250
Dehydration ethanol/acetone*'*’ 50*, 75*, 90*, 100*, 100'‘”, 100‘“’ 10 min each (100 x2) 40 sec each 250
Infiltration Procure Araldite 5, 20, 50, 60, 75, 80, 90, 100* 4-8 h each 3 min each 250
(100 x2 (v)
)
Polymerization Procure Araldite 100* conventional oven 60 °C overnight
Note: Concentrations in bold represent the microwave shecule, concentrations not in bold represent the conventional schedule, and * represents
concentrations used in both schedules.
(v) = steps undertaken in vacuum
on/offdenotes magnetron (irradiation) cycle
= acetone
Preparation of radulae from adultanimals
For samples processed using either conventional or mi-
crowave-assisted methods, the dissected tissue mass was im-
mediately immersed in a fixative comprised of2.5% glutar-
aldehyde buffered in 0.1 M phosphate, with a pH of7.2 and
an osmolarity of 900 mmol kg"' adjusted using sucrose
(buffer A). The buccal mass and radula were separated from
the remainder of the animal, and the radula was either left
whole or cut transversely into three or four segments (the
buccal mass was discarded). The tissues were then processed
by either conventional bench-top methods or accelerated
microwave-assisted protocols using a Pelco Biowave® fitted
with a cold spot and vacuum chamber, according to the
specific schedules detailed in Table 1. This included fixation
with glutaraldehyde in buffer A, rinsing in buffer A, post
fixation in 1% osmium tetroxide (OSO4) in 0.05 M phos-
phate buffered saline (buffer B) at 4 °C, and a final rinse in
buffer B, prior to dehydration through a graded series of
acetones, then infiltration and embedding in Spurr’s resin.
Preparation of radula tissue by high-pressure freezing was
not possible for adults due to limitations in sample size,
which was again restricted to -200 pm.
Following polymerization, resin blocks were sectioned
for observation at both the LM and TEM level. For LM, 1
pm-thick sections were mounted on glass slides and stained
with ac]ueous 1% Methylene Blue and 1% Azur II (20 sec)
prior to imaging on an Olympus BX51 optical microscope
Figure 2. Light micrograph of a longitudinal section through a fitted with an Olympus DP70 digital camera. For TEM, ~60
major lateral tooth from adultAcanthopleura hirtosa at row 6 pre- nm-thick sections were mounted on copper grids and
pared using microwave-assisted protocols. Despite being situated
double stained with uranyl nitrate (single crystal in one drop
deep within the tooth stylus (ts), the tissues ofthe stylus canal (sc) of50% methanol 10 min) followed bySato’s modified lead
are well preserved, se, superior epithelium; tc, tooth cusp. Scale ) (
bar = 50 pm. citrate (10 min) (Hanaichi et al. 1986) prior to imaging on
a lEOL 2000 TEM at 80 kV.
38 AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008
methods were used, microwave-
assisted protocols dramatically re-
duced sample processing times from
six days to one hour (Table 1) and re-
sulted in superior ultrastructural pres-
TEM
ervation at the level. In addition,
microwave-assisted protocols in-
creased fixative penetration into the
tooth, improving, for example, tissue
preservation within the stylus canal
(fig. 2), which fixedpoorlybyconven-
tional methods.
The endothelial radula sheath
layer, recognizable by the presence of
ciliated endothelial cells distributed
over the entire sheath’s surface, re-
Figure 3. Major lateral tooth rows 12-14 from radulae of adult Acanthopleiira hirtosa pre-
mained intact when processed by mi-
pared using (A) conventional and (B) microwavetechnic]ues. Note the separation (arrow) of
the superior epithelium from the anterior face (af) ofthe tooth cusps (tc) in conventionally crowave methods (Fig. 1), in contrast
fixed material and the poor infiltration of resin into the tooth stylus (ts) and cusp in to conventional methods where it was
microwave prepared material. Scale bars = 200 pm. often disrupted. It is likely that the
shorter sample processing and han-
dling times with microwave fixation
Preparation of radulae from juvenileanimals reduce the likelihood ofdamage to this delicate membrane.
Juvenile specimens ofAcanthopleiira hirtosa were pro- Both techniques preserved prominent vesicles that line the
cessed similarly using conventional and microwave-assisted anterior and posterior surfaces of the tooth cusps (Fig. 3).
methods; however, schedules were adjusted in order to ac- We have found that these vesicles can be either abundant or
count for the reduction in tissue size, and samples were virtually absent at the same stage of tooth development in
embedded in Procure Araldite (formerly Epon Araldite) different animals. It is currently not known whether these
(Table 2). Eor cryo-fixation ofjuvenile radulae, the imma- vesicles are natural features ofthe adult epithelium or arti-
ture portion of each radula was removed by cutting trans- facts resulting from the fixation process.
verselyapprox. 200 pm from the posterior end. Each imma- Conventional fixation often resulted inthe separation of
ture portion was then placed into a 200 pm membrane filled the superior epithelium from the hard tooth cusps, while in
with 20% bovine serum albumin in artificial seawater. These microwave-fixed material this artifact was rarely observed
membrane-mounted samples were rapidly frozen in a high- (Fig. 3). Despite a number of attempts to improve resin
pressure freezer (Leica EMPACT 2) prior to cryo- penetration into the base material using both conventional
substitution in acetonecontaining2% OSO4at -85 °C for 52 and microwave-assisted methods, includinglower resin con-
h. Samples were then progressively warmed from -85 °C to centrations and increased infiltration times, the fibrous ap-
20 °C over 13 h in a Leica automatic freeze substitution unit pearance ofthe majorlateral tooth cusps andstyluspersisted
prior to beingwashed in acetone and infiltrated and embed- and was indeed far more noticeable in microwave-prepared
ded in Araldite resin. Cryo-prepared sample blocks were specimens (Fig. 3). This is indicative ofpoor infiltration by
polymerized underwater using a Pelco Biowave® microwave the epoxy resin and is a common problem encountered in
W
at 650 for 90 min. Conventional, microwave-assisted and mollusc species by many researchers (Nesson and Lowen-
cryo-prepared sections were imaged unstained on a JEOL stam 1985, Mackenstedt and Markel 1987).
2100 TEM at 120 kV, using a Gatan Orius SC1000 digital Microwave-assisted sample processing resulted in far
camera. better preservation oftissue ultrastructure comparedto con-
ventional methods. TEM of both conventional and micro-
wave-prepared samples revealed the typical arrangement of
RESULTS AND DISCUSSION organelles within the superior epithelium near the cusp sur-
face that are common to chitons, including microvilli, mi-
Preservation of adult radulae tochondria, rough and smooth endoplasmic reticulum, and
While glutaraldehyde fixation ofradula epithelium was abundant, electron-dense ferritin siderosomes (Fig. 4).
satisfactory at the LM level when conventional bench-top However, at higher magnifications it could be clearly seen
SAMPLE PREPARATION OF RADULA EPITHELIUM 39
sue from the tooth cusps and bases,
high-pressure freezing (HPF) resulted
in unsurpassed preservation of juve-
nileAotnOop/enra hirtosa radula tissue
(Fig. 6C). Nuclei (with well defined
chromatin adjacent to the nuclear
membrane), mitochondria (with well
preserved cristae), rough endoplasmic
reticulum, and Golgi apparatus were
clearly represented in the cytoplasm,
together with microvilli and ferritin
siderosomes (Fig. 6C). The ~60 nm
granules observed in microwave-
assisted preparations were also present
in HPF samples (data not shown). The
large vesicles surrounding the tooth
cusps in adult epitheliumwere also ob-
Figure4. Transmission electron micrographs ofthe radula superior epithelium abutting the served in juvenile tissue prepared us-
tooth cusp (tc) of (A) conventionally and (B) microwave-processed radulae from adult ing conventional and microwave pro-
Acantliopleiira hirtosaattooth rows 14and 13, respectively, f,ferritin siderosome;g,granules; tocolsbut were absent in HPF material
mt, mitochondria; mv, microvilli. Scale bars = 5 pm. (data not shown). While variations in
that samples fixed usingthe microwavecontained numerous
granules approx. 60 nm in diameter. These granules, which
are likely to be either aggregations ofribosomal material or
glycogen, appeared throughout the cytoplasm, particularly
near the apical poles of the superior epithelium (Fig. 5).
These fine structures are either absent or not well preserved
in conventional preparations and have not been reported by
previous authors (Nesson and Lowenstam 1985, Kim et al.
1989). It is likely that the retention of these structures in
microwave-prepared samples is a result of the dramatic re-
duction in processing time of samples, thereby reducing
chemical exposure andthechance ofextractingsolublecom-
ponents of the tissue.
Preservation ofjuvenile radulae
The type and arrangement oforganelles within the api-
cal epithelium ofjuvenile Acanthopleiim hirtosa radulae fol-
lows the same characteristic configuration as that described
for adult tissue. The preservation equality of juvenile radula
epithelium, when using conventional and microwave-
assisted methods, was also very similar to that observed in
adults (Figs. 6A-B). While the reduction in sample size may
improve fixative penetration in juvenile radulae, the com-
parable fixation equality between adult and juvenile tissue Figure 5. Transmission electron micrograph of (A) conventional
indicates that size is not a limiting factor. In support ofthis, and (B) microwave-preparedsuperiorepithelium from adultAcan-
the -60 nm granules were absent in conventionally fixed thopleura hirtosa showing differences in the preservation of fine
structure. Arrows denote aggregations of ribosomal- or glycogen-
juvenile tissue but were retained in adult and juvenile tissue
when processed by the microwave protocol (Figs. 5B, 6B). bliakress=tructpumr.es within the cytoplasm, f, ferritin siderosome. Scale
With the exception ofslight separation ofepithelial tis- 1
40 AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008
juveniles or very small mollusc species
can be prepared using this method. In
addition, the relative portability and
affordability of microwave technology
compared to HPF makes it a more re-
alistic option for many laboratories.
While each of these new techniques
has proven to be suitable for fixation
oftissue at the LM level, the improved
retention of ultrastructural informa-
tion gained by using microwave and
HPF methods highlights the need for a
re-evaluation of fine cell structure in
molluscan radulae.
While the ultrastructure of the
radula epithelium ofchitons and lim-
pets has been well documented (Nes-
son and Lowenstam 1985, Mann et al.
1986, Kim etal. 1989, Rinkevich 1993),
no studies havebeen conducted on the
development of this tissue as teeth
progress from an unmineralized to a
mineralized state, information that is
crucial for resolving the cellular basis
of biomineralization. In addition,
cryo-techniques have recently been
used in conjunction with chemical
fixation to characterize the organic
matrix in limpets, by dramatically
reducing artifacts resulting from stain-
ing, dehydration, and embedding
(Sone et al. 2007). The high-pressure
freezing method outlined in the
Figure 6. Transmission electron micrographs comparing the preservation ofcell ultrastruc- current study therefore provides a
ture in radula epithelium from juvenile specimens ofthe chiton Acmithopleura hirtosa pro-
valuable first step in preserving
cessed using (A) conventional, (B) microwave-assisted, and (C) high-pressure freezing pro-
the organic matrix for subsequent
tocols. Black arrows denote aggregations ofribosomes/glycogen, f, ferritin siderosome; mv,
cryo-sectioning in a frozen hydrated
microvilli; mt, mitochondria; n, nucleus; tc, tooth cusp. Note: all imagesweretaken from the
first tooth row after the onset ofmineralization in the cusps. Scale bars == 2 pm (images on state. The methods ofsample prepara-
left) and 1 pm (images on right). tion presented here not only will ben-
efit future investigations of the supe-
rior epithelium and organic matrix of
ultrastructure may arise from differences in the functional chitonsand limpetsbut also will beofusefortaxonomic and
state of the cells at the time of fixation (Hayat 2000), it is morphological studies ofmolluscan radulae in general.
more likely that these vesicles are artifacts resulting from
ACKNOWLEDGMENTS
glutaraldehyde fixation (Bowers and Maser 1988).
Despite a slight improvement in resin infiltration using The authors would like to thank the assistance of Mr.
the conventional method, microwave-assisted processing of Gordon Thompson at Murdoch University for his advice on
radula superior epithelium is preferred due to the improved histological preparations. This research wascarriedoutusing
qualityoftissue preservation and the vastly reduced time for facilities at The Centre for Microscopy, Characterisation and
sample processing. While cryo-preservation using HPF re- Analysis at The University of Western Australia and The
sults in excellent ultrastructural preservation, it is limited Centre for Microscopy and Microanalysis at The University
with respect to sample size. As such, only the radulae of of Queensland, both of which are supported by university.
SAMPLE PREPARATION OP RADULA EPITHELIUM 41
state, and federal government funding. Pinancial support McDonald, K. L., M. Morphew, P. Verkade, and T. Mtiller-
was provided by The Australian Research Council (Grant # Reichert. 2007. Recent advances in high-pressure freezing:
DP0559858). We also acknowledge the technical, scientific, Equipment and specimen loading methods. In: ]. Kuo, ed..
and financial assistance from the NANO-MNRP. All experi- Electron Microscopy Methods and Protocols. Humana Press,
ments conducted during the study comply with the Austra- Totowa, New Jersey. Pp. 143-173.
lian Code of Practice for the Care and Use of Animals for Nesson, M. FI. and H. A. Lowenstam. 1985. Biomineralization pro-
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